1. Field of the Invention
The present invention generally relates cellular therapy and tissue engineering for treating atherosclerosis and heart failure, and more particularly to the synthesis and expression of clusterin (apolipoprotein-J) and its use in cellular therapy and tissue engineering, especially for protecting embryonic and adult stem cells against inflammatory injury and apoptosis.
2. Description of Related Art
Cellular therapy with multipotent stem cells for treatment of myocardial infarction and ischemic heart failure. During the development of ischemic heart failure caused by disruption of coronary circulation, progressive loss of functional myocytes occurs in the myocardium. Since adult mature myocytes do not proliferate, the damaged myocardium is replaced by connective tissue composed of non-cardiomyocytic cells and extracellular matrix and a scar surrounded by hypertrophied myocytes. By contrast, fetal cardiac myoblasts or stem cells can proliferate and differentiate into adult myocytes. Fetal myogenic stem cell proliferation remains active until the second week after birth. Recent studies have suggested that implantation of fetal cardiac or skeletal myoblasts1-4 may help heal an experimentally infarcted heart. These studies show that when implanted in the infarcted adult heart, embryonic myoblasts may survive, proliferate, and communicate with host cells in the myocardium. Using a swine model, van Meter, et al.6,7 reported that transplanted myoblasts formed close associations with host myocytes that resembled nascent intercalated disks on electron microscopy. These cells also contained myofibrils and other cell architecture resembling the transplanted cell lines, and they may exert an angiogenic influence resulting in the proliferation of the surrounding microvasculature.
In spite of the rapid progress in the studies of myocyte implantation, there is little information concerning the differentiation of the implanted fetal myocytes in the heart. The cell lineages in the heart, including cardiac myocytes, vascular cells and interstitial cells, are derived from embryonic stem cells. In addition to highly mature cardiac myocytes, some embryonic myoblasts, in particular those located in subepicardium, may differentiate into coronary arterial cells. It is now accepted that coronary arteries form originally in the subepicardial area and subsequently grow into the aorta. During embryonic development, the first signs of coronary vessel formation appear as blood island-like structures or endothelial tubes in the subepicardium.
In addition to fetal or embryonic tissues, adult or new-borne tissues may contain certain numbers of stem cells that differentiate into mature, functional cells in different organs. One of the major resources for adult stem cells is the bone marrow. Many studies recently have shown that stem cells from both human and animal bone marrows can differentiate into myocyte-like cells.1,4 Transplantation of bone marrow stem cells into the heart with experimental infarction leads to development of neomyocardial and neovascular tissues as well as improvement of heart function.
U.S. Pat. No. 6,805,860 (Alt) describes a process for repairing tissue of a patient's heart, which comprises delivering stem cells, preferably autologous stem cells, to the site of tissue to be repaired. The stem cells are injected through a catheter to invade the failing tissue at the site, while local forces at the site are quelled from disrupting migration of the stem cells into the failing tissue.
U.S. Pat. No. 6,607,720 (Xiao, et al.) describes a therapeutic method for improving cardiac function after myocardial infarction using genetically altered mammalian embryonic stem cells. Exemplary cDNAs said to be useful for transfection are VEGF (vascular endothelial growth factor); FGF1,2(fibroblast growth factor 1 and 2); TGF-α & β1-5 (transforming growth factor α & β1-5; IGF-1 & -2 (insulin-like growth factor 1 & 2); SERCA I & II (sarco/endoplasmic reticulum Ca2+ ATPase I & II); β2 (beta adrenergic receptor II); Gs-protein (stimulatory guanosine-binding protein); Ca.2+ channel (calcium channel); and telomerase.
Because of the limited supply of autologous stem cells, most studies use allogenic stem cells for transplantation. In general, stem cells are weak antigens that evoke little immune reaction. However, allogenic stem cells are transplanted frequently into the heart with infarction where they confront a very harsh environment. Local inflammation, oxidative stress and cytotoxic radicals and proteins may cause death of the transplanted cells primarily via an apoptotic mechanism. Even in the weak immune reaction to stem cells, long-term exposure to activate immune cells and their cytokine products may also trigger cell death by apoptosis. Apoptosis is a form of genetically programmed cell death that represents a major mechanism by which tissue removes unwanted, aged or damaged cells under both physiological and pathological conditions. Morphologically, apoptosis is characterized by chromatin compaction and margination, by nuclear condensation and fragmentation, and by cell shrinkage and blebbing.
Apoptosis may occur abnormally leading to accelerated cardiac cell death during heart failure, as demonstrated in animal models.8 During the development of atherosclerosis, accumulating free cholesterol undergoes oxidation, producing oxysterols with higher cytotoxicity to vascular cells. In vitro studies have shown that some oxysterols such as 7-ketocholesterol exerts potent apoptotic effects on stem cells. Oxysterols are considered to be major cytotoxic components of oxidized low density lipoprotein (oxLDL). It has been shown that CD95 is present in human plaque, and it was proposed that activation of CD95 may mediate apoptosis of stem cells in the presence of IFNγ and TNFα. (Geng et al, Arterioscler Thromb Vasc Biol. (1997) 17:2200-8). The role for apoptosis in the development of neocardiovascular tissues remains unclear, however.
Clusterin (Apolipoprotein-J)12 is a sulfated, heterodimeric glycoprotein containing two 40 kDa chains joined by a unique five disulfide bond motif, as schematically illustrated in FIG. 1. Encoded on a 2-kb mRNA, clusterin is transcribed from a single copy gene located on mouse chromosome 14.13 It contains several domains, such as amphipathic helix, heparin-binding domain, and lipid-binding domain. This protein was initially identified from ram rete testes fluid and named for its ability to elicit clustering of Sertoli cells supporting sperm maturation and development (NCBI/GenBank Accession No._NM—203339, NM—001831) Thereafter, species homologues have been isolated and cloned by a number of groups working in widely divergent areas, resulting in a number of synonyms including testosterone repressed prostate message-2 (TRPM-2), sulfated glycoprotein-2 (SGP-2), apolipoprotein-J (clusterin), SP-40, 40, complement cytolysis inhibitor (CLI), and dimeric acidic glycoprotein (DAG), gp 80, NA1/NA2, glycoprotein III, etc. Clusterin is constitutively expressed by various tissues and cells, in virtually all body fluids, and on the surface of cells lining body cavities. It circulates in blood with the high density lipoprotein (HDL) fractions, and thus considered as a component of HDL in which clusterin is associated with apolipoprotein-AI and paraoxonase (NCBI Accession No. NM—000446). The latter protein is one of the key enzymes with antioxidant property. Clusterin and its associated proteins are present at high levels in the lesions of patients with atherosclerosis.25 Clusterin is translated as a typical hydrophobic signal peptide with 21 amino acids in length.14 The biological functions of clusterin have not been completely known. Reported functions of clusterin include apoptosis regulation, complement defense, lipid recycling, membrane protection, and maintenance of cell-cell or cell-substratum contacts. It can effectively bind to lipids including both cholesterol and oxysterols, and has been shown to promote efflux of cholesterol and oxysterols from lipid-laden foam cells. This protein can also inhibit complement-mediated cell death, and promote cell aggregation and adhesion. Recently, clusterin has been found to be an anti-apoptotic protein. It has been reported that clusterin expression is induced and confers resistance to apoptotic cell death induced by heat shock and oxidative stress. High levels of clusterin have been shown in tissues with apoptosis. However, careful analysis of the producing cells revealed that clusterin expression is restricted to the vital cells adjacent to dead cells, suggesting that this molecule may act as a cell survival factor, which protects bystander cells. Recent studies have shown that clusterin is an anti-apoptotic protein.15 
Developmental regulation of clusterin expression has been reported in many tissues including the heart, kidney, lung, and brain.16,17 In the heart, clusterin is found in both the atria and ventricles of the fetal mouse heart, but in the adult heart, only the atria show positive stains for clusterin.13 However, marked induction of clusterin can be detectable in the heart with acute infarction18, in particular the peri-infarct zone.19 Induction of clusterin is also observed in the myocardium with inflammation,20 suggesting a protective effect of clusterin in the inflammatory myocardium. Clusterin-deficient mice appear to be more sensitive to develop myocarditis than age-and sex-matched wild type controls. In the kidney, clusterin is expressed in the ureteric bud but not in surrounding mesenchyme.21 When the mesenchyme is induced to differentiate into renal epithelium, clusterin expression takes place and continues in developing tubules. In newborn mice, almost all the tubules express clusterin, but adult tubules rarely express clusterin. Similar to the time course in the heart and kidney, the developing fetal but not adult lung contains clusterin.
The temporal expression of clusterin during ontogeny and tissue injury implies a role for clusterin in organogenesis and tissue remodeling, perhaps through regulation of stem cell proliferation, differentiation and apoptosis, and interactions with other cellular components or extracellular matrix.16 Little and Mirkes22 recently investigated the relationship between clusterin expression, normal programmed cell death (PCD) in the developing rat limb bud, and abnormal cell death induced by hyperthermia in day 11 rat embryos. They observed that clusterin mRNA and protein were expressed at high levels in the heart, a tissue that is completely resistant to the cytotoxic effects of hyperthermia. Similar finding occurs in the developing brain. Clusterin expression occurs in the earliest neurons of the cortical plate on embryonic day (E) 12, and can continue to increase in an age-dependent manner, with the greatest intensity of expression being found in the postnatal mature brain.23 Clusterin is also frequently found in neuron degenerative disorders, such as Alzheimer's disease.24 
In order to better implement the full potential of stem cell transplantation for treatment of atherosclerosis, myocardial infarction and heart failure, new ways are needed to promote or enhance the success of cellular therapy, including improving the survival of transplanted cells.